(1) Field of the Invention
The present invention relates to a method for determining lipids using thin layer chromatography (TLC) to separate the lipids and then multiple enzyme catalyzed steps culminating in chromogen staining of the TLC separated lipids. In particular, the present invention relates to a method which uses a hydrolase enzyme, an oxidase enzyme and a peroxidase enzyme in the presence of a chromogen precursor to form an oxidized chromogen stain.
(2) Prior Art
Lipids are fatty acid esters and particularly include triesters of glycerol. Phospholipids are lipids characterized by the formula: ##STR1## where m and n are an integer between 10 and 16 and R is selected from various groups. Lipids include cholesterol, lecithin, cephalin and the like. The compounds are characterized by diester linkages and a phosphate group. The determination of lipids, particularly phospholipids, in human and animal tissue and serum is important in relation to neoplastic disorders, diabetes mellitus, arteriosclerosis, coronary heart disease, extra hepatic obstruction detection and amniotic fluid analysis.
Conventional electrophoretic systems separate molecules by charge and molecular weight. However, when the molecular weight and charges of the molecules are similar, alternate fractionation procedures must be used. Thin layer chromatography (TLC) can separate the components in a mixture based on their polarity. Phospholipids, naturally occur as a heterogenous mixture of individual compounds of variable polarity whose molecular weights are similar and usually less than 1000 and are particularly difficult to identify. TLC has provided a tool to separate and quantitate phospholipids as well as other lipids.
Current methods of TLC phospholipid quantitation employ charring with sulfuric acid (Gluck, L., et al. Am. J. Obstet Gynecol Ref 120,142-155 (1974)), chemical determination of the phosphate groups (Kolins, M. D., et al, Clin. Chem. 26, 403-405 (1980)) and determination with anagolene sodium, i.e. Coomassie dye (Nakamura, K., and Handa, S., Anal. Biochem. 142, 406-410 (1984)). None of these staining procedures are specific for the phospholipids. Furthermore, phospholipid determination by sulfuric acid presents inherent quantification problems due to variable charring of the phospholipid classes (Gluck, L., et al, Clin. Perinatol 1, 125-131 (1974)). Recently, enzymic procedures for the determination of lecithin, sphingomyelin and phosphatidylglycerol in aqueous solution have been developed. McGowan, M. W., et al, J. Clin. Chem. Clin Biochem, 20 807-812 (1982); McGowan, M. W., et al, Clinical Chemistry 29 1513-1517 (1983); Artiss J. D., et al Clinical Chem 30 534-537 (1984); McGowan, M. W., et al, Microchemical Journal 31 216-223 (1985); Bozinowski, D. et al Microchemical Journal 32, 254-265 (1985); Artiss, J. D. Annals of Clin. and Lab Science 15 488-494 (1985). This procedure allows only a single determination in a given solution.
Other prior art includes Han J. et al, Korean J. Biochem 17(1) 19-30 (1985); Higashi, H, Seikagaku 57(2) 133-135 (1985) and Ambrosetti, P., Chromatographic 9(12) 633-634 (1976). The Ambrosetti reference describes bromine oxidized phosphate esters which inhibit enzyme activity. The stained areas are separate from the oxidized phosphate esters where enzyme activity is not inhibited. Bromine is difficult to handle in the laboratory. Higashi uses antibodies. Han describes TLC and an enzymatic assay with an enzyme attached to an antibody label. None of the procedures are regarded as being quantitative.